Recombinant DNA techniques are currently being used to create genetically engineered cells capable of producing large quantities of a desired protein. It has been found that protein produced by genetically engineered cells can accumulate in an insoluble form which is difficult to extract by conventional techniques. A need exists for a commercially practical method capable of extracting this insoluble form of protein.
Fraenkel-Conrat, Virology, 4:1-4 (1957) discloses a method for degrading tobacco mosaic virus (TMV) with acetic acid. The reference discloses that cold 67% acetic acid splits TMV and causes precipitation of the nucleic acid from the solution. Two volumes of glacial acetic acid cooled to just above its freezing point are added to a cold virus solution. The resulting solution is cooled to about 3.degree. C. and a precipitate appears, which is removed by centrifugation after 15 minutes. Native protein free from nucleic acid or other gross contaminants can be isolated from the supernatant by dialysis.
U.S. Pat. No. 4,364,863, issued to Leibowitz et al., discloses a method of extracting leucocyte and fibroblast interferons from interferon-expressing bacterial cells. The disclosed method comprises acidifying a suspension of interferon-containing bacterial cells, removing substantially all of the suspension liquid from the cells, preparing a second suspension of the acidified cells, neutralizing the second suspension, separating the interferon containing liquid from the suspended cells, and isolating the interferon from the liquid. The patent discloses that suitable acids are hydrochloric acid, nitric acid, sulfuric acid and phosphoric acid. The extraction is conducted at a temperature of from about ambient temperatures to about 40.degree. C.
U.S. Pat. No. 4,405,601, issued to McEntire et al., discloses a method for the extraction and purification of biologically active lymphokines from large scale culture supernatants. The method comprises effecting the growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines and thereafter harvesting, concentrating and clarifying the resulting supernatant culture fluids. The supernatant culture fluids are extracted with a solvent selected from the group consisting of trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate and perchloric acid and removing the solvent to produce a lymphokine fraction which exhibits the capability of inducing cell-mediated immunity reactions and tumor regression in mammals.
U.S. Pat. No. 4,450,103, issued to Konrad et al., discloses a process for recovering human IFN-beta (fibroblast interferon) from transformed bacteria. The process comprises disrupting the cell membranes of bacteria; solubilizing the IFN-beta from the disruptate into an aqueous medium with a solubilizing agent such as sodium dodecyl sulfate; extracting the IFN-beta from the aqueous medium with 2-butanol, 2-methyl-butanol, or mixtures thereof under conditions that maintain phase separation between aqueous medium and the extractant; and isolating the IFN-beta from the extractant such as by precipitating the IFN-beta from an aqueous buffer mixture of the extractant by lowering the pH thereof.